Multi-stage nutrigenomic diagnostic food sensitivity testing in animals

ABSTRACT

A multi-stage method for diagnosing an immunologic food sensitivity or intolerance in a companion animal. Firstly a saliva or blood spot or other non-serum bodily fluid sample is collected. The screening the saliva or blood spot or other non-serum bodily fluid sample detects the presence of at least one of IgA or IgM antibody to a particular food ingredient or composition. An immunologic food sensitivity or intolerance based on the presence of the antibody is diagnosed. Secondly a blood sample is collected and serum from the sample is screened to detect the semi-quantitative or quantitative presence of at least one of an IgA, IgM or IgG antibody or immune complex to a particular food ingredient or composition. An immunologic food sensitivity or intolerance based on the presence of the antibody or immune complex is diagnosed. Thirdly, a biologically active nutrient in relation to the animal from a molecular dietary signature is determined. The molecular dietary signature for the animal is a variation of expression of a set of genes, proteins or metabolites which may differ for the genotype of each animal.

RELATED APPLICATIONS

This application is a Continuation in Part of MULTI-STAGE NUTRIGENOMICDIAGNOSTIC FOOD SENSITIVITY TESTING IN ANIMALS (Dodds) Ser. No.12/545,041 filed Aug. 20, 2009, and which claims the benefit of priorityof earlier filed Us applications. This application is also aContinuation-in-Part of application Ser. No. 12/465,603, filed May 13,2009, entitled FOOD SENSITIVITY TESTING IN ANIMALS (Dodds), which claimsthe benefit of and priority to U.S. Provisional Application Ser. No.61/147,443, filed Jan. 26, 2009, entitled FOOD SENSITIVITY TESTING INANIMALS (Dodds). This application is concerned with and relates to thedisclosure of application Ser. No. 12/316,824, filed Dec. 16, 2008,entitled DIAGNOSTIC SYSTEM FOR SELECTING NUTRITION AND PHARMACOLOGICALPRODUCTS FOR ANIMALS. The contents of all of these applications areincorporated by reference herein in their entirety.

BACKGROUND

The disclosure is directed to food sensitivity testing in companionanimals. This disclosure is also concerned with animal nutritiondiagnosis. More particularly the disclosure is directed to the testingand diagnosis of genetic issues relating to nutrition issues anddisorders of companion animals, for instance dogs and cats.

More specifically the disclosure is directed to animal nutritiondiagnosis and care, animal well-being and nutrition, and methods andsystems for enhanced determination of these factors.

A common health concern identified by health surveys of several purebreddog clubs is food sensitivity or intolerance. Other than time-consumingfeeding trials, which eliminate potential allergic ingredients everyseveral weeks, testing for this disorder uses expensive and unsightlyskin patch testing or serum allergy screening that lack specificity.

Delayed food sensitivities in people are extremely common and can bemanifested by gastrointestinal, neurological, pulmonary, dermatologic,ear, nose and throat, musculoskeletal, genitourinary, cardiovascular andendocrine problems. Similar clinical problems are manifested in animalswith food sensitivities.

Diagnostic testing systems available for humans are typically based oneither immunoglobulin E (IgE) or immunoglobulin A (IgA) or a combinationof immunoglobulin G (IgG) antibody or immune complex testing mediated bycomplement.

The newer test methodologies for humans are run on serum, feces, orsaliva and typically use ELISA or other immunoassay platforms such aslateral flow, or latex or bead agglutination, and identify IgG or IgA orimmune complex reactions to food ingredients that are mediated bycomplement, as well as IgA or immunoglobulin M (IgM) antibodies to foodingredients that are elaborated in saliva.

Research has shown that the key to delayed or latent or pre-clinicalfood sensitivity testing in humans is the identification of theoffending IgG or IgA antibodies and immune complexes in serum or feces,and the offending IgA or IgM antibodies in saliva. In fact, antibodiesto food ingredients can appear in the saliva before the clinical orgastrointestinal biopsy diagnosis of inflammatory bowel disease or“leaky gut syndrome” is made in human patients. Saliva testing can thusreveal the latent or pre-clinical form of food sensitivity. A similarelaboration of IgA or IgM antibody in saliva but not serum pertains toanimals with latent or pre-clinical gastrointestinal disease.

Delayed sensitivities in humans are usually revealed as soon as 2 hoursor as long as 72 hours after eating, which is the reason it can bedifficult to connect the symptoms with a food or foods eaten as long asseveral days previously. There is a very high correlation betweendelayed food sensitivity and the amount and frequency of the foodconsumed.

In human serum testing, food sensitivity reactions in the gut lead toincreased blood levels of IgA or IgG directed to these food ingredients.Similarly, the immune complexes being formed from food reactions in theblood adhere to red blood cells and these altered blood cells are thencleared by the body's recticuloendothelial system in the liver andspleen. Individuals having more immune complex on their red blood cellsare the ones who suffer from chronic food sensitivities.

In saliva testing, deposition of food antigens or peptides in the guthas been documented in humans to lead to the production of IgA or IgMantibodies in the serum and in secretions such as saliva. In somesituations, IgA or IgM antibodies to food ingredients appear in salivabut are not present in serum. So in humans salivary antibodies serve asan indication of a general mucosal immune response and can be induced inpeople and animals without parallel antibodies being detected in serum.

There is a need to provide for practical and rapid screening or testingfor food sensitivity and intolerance to permit enhancement of the healthof animals.

Studies have indicated that specialized nutrient intake extends andimproves life, delays onset and slows progression of disease, andenhances the quality of life of companion animals.

Changing the proportions of macro-nutrients and micro-nutrients indifferent nutrient and food products is important in obtaining the rightbalance. To date, the utility of such characteristics and components hasbeen limited or not as useful as possible.

Currently, time consuming elimination dietary trials are done where oneingredient at a time is removed and the remaining diet is fed for six toeight weeks to determine if the animal patient's food sensitive or foodintolerance symptoms subside. Alternatively, arbitrary selection can bemade of a food preparation containing limited, namely restricted antigensource, or novel, namely not fed previously, ingredients(s) areemployed. Both these techniques are imprecise or indirect methods ofaddressing the problem.

The present disclosure provides for screening or testing animal subjectsfor sensitivity or intolerance relative to dietary compositions, and thetesting and screening should be advantageous and commercially useful.

There is also a need to conduct periodic comprehensive nutritionassessments of animals.

The probability that an individual animal will develop a specificnutrition-related condition in its lifetime is a product of complexinteractions between its genetic makeup, environmental influencesincluding diet, and agents of disease (e.g., chemical, physical, orbiological) that the animal encounters.

More specifically, it is necessary in animal nutrition diagnosis andcare for comprehensive nutrition assessments of animals for diseases anddisorders of animals be achieved in order to reduce morbidity andmortality, and improve the quality of life and lifespan.

It is important for nutritionists or other animal food professionals toprescribe or recommend nutrient needs or diets on the basis of moreprecise knowledge of how nutrients or food components interact at thelevel of the genome, where these constituents act by “up- ordown-regulating” a set of target genes. Animal nutritionists or otheranimal food professionals should design nutrients or foods tailored tothe genome or genomic profile or to prescribe or recommend the inclusionof specific molecules in the diets of animals to optimize physiologicalhomeostasis, disease prevention and treatment, and productive orreproductive performances. Individualized nutrition requires an evenmore refined technique or approach than is currently available orapplied.

SUMMARY

There is provided a multi-stage nutrigenomic diagnostic test system forscreening or testing for food sensitivity or intolerance in animals, inparticular companion animals, such as dogs, cats, rabbits, hamsters, andhorses.

In particular there is a system for providing staged testing from afirst test protocol, being saliva or other mucosal fluid based test,followed by a serum based test protocol and then a genetic DNA/RNAnutrigenomic test protocol. In other cases there is a system forproviding staged testing from a first test protocol, being a serum basedtest protocol and then a DNA/RNA nutrigenomic test protocol. In yetfurther cases there is a system for providing staged testing from afirst test protocol, being a saliva based test, followed by a DNA/RNAnutrigenomic test protocol.

A multi-stage method for diagnosing an immunologic food sensitivity orintolerance in a companion animal. Firstly saliva or other non-serumbodily fluid sample is collected. The screening the saliva or othernon-serum bodily fluid sample detects the presence of at least one ofIgA or IgM antibody to a particular food ingredient or composition. Animmunologic food sensitivity or intolerance based on the presence of theantibody is diagnosed. Secondly a blood sample is collected and serumfrom the sample is screened to detect the quantitative presence of atleast one of an IgA, IgM or IgG antibody or immune complex to aparticular food ingredient or composition. An immunologic foodsensitivity or intolerance based on the presence of the antibody orimmune complex is diagnosed. Thirdly a biologically active nutrient inrelation to the animal from a molecular dietary signature is determined.The molecular dietary signature for the animal is a variation ofexpression of a set of genes, proteins or metabolites which may differfor the genotype of each animal.

Immunglobulins in companion animals differ from those in humans incertain structural and functional aspects in blood, body fluids andtissues. Specifically, cats have significantly more IgA in saliva andserum than dogs or humans, although dogs also have less serum IgA thanhumans. Levels of IgA and IgM are correlated in dog saliva and tears,but there are conflicting data regarding the correlation between serumand secretary IgA levels in dogs. Further, cats with oral diseases suchas gingivitis or stomatitis have increased levels of salivary IgG andIgM but less salivary IgA, whereas serum levels of all three of theimmunoglobulins are uniformly increased. Salivary immunoglobulin levelsin cats remain relatively constant whereas those of dogs vary from dayto day and there may be diurnal variation with higher levels in theafternoon.

Similarly, the salivary and serum IgM levels of dogs and cats are higherin amounts from those of humans.

Saliva can be used as a diagnostic tool to assess the health or diseasestatus of an animal. Saliva is easily collected, stored and shipped, andprovides a non-invasive means of multiple or serial sampling for use asa diagnostic tool for a variety of conditions in animals.

The measurement of selected blood (serum) and salivary antibodies iscompared in healthy individual animals and in those animals known tohave or suspected of having food sensitivities or intolerances. Thereliability of saliva testing depends on knowing the correlation ordifferences between the salivary and blood concentrations, activity, orconstituents of a particular substance. The transfer of substances fromblood into the saliva or vice versa is dependent on their physiochemicalproperties. Faster transfer rates of molecules are associated with smallmolecular weight and great lipid solubility.

A good correlation exists between the saliva/blood ratio of substancesand salivary pH. Salivary flow rate and any existing pathophysiology ofthe oral cavity have also been shown to affect salivary distribution ofsubstances. Saliva content of antigens and antibodies reflects thenutritional and metabolic status of the body, as well as the metabolic,hormonal, biochemical, physiological, immunological or even emotional,status of the individual animal.

Food sensitivity testing for common offending allergens and peptides indogs or cats is achieved. The sensitivity and testing is for grains mostoften associated with inflammatory bowel disease (“leaky-gut syndrome,intestinal dysbiosis) or and other symptoms of adverse foodreactions—such as, but not limited to wheat and other glutens, corn andsoy. These three grain types are among the major constituents (top 5ingredients) that make up the bulk of standard commercial pre-processedand pre-compounded kibble fed to most dogs or cats. Another commonallergen in pet foods or animal food compositions is beef, and thetesting and screening is also directed to but not limited to othermeats, fish, dairy, eggs, other grains, botanicals, oils from seeds orfish, botanicals, vegetables, or fruit.

The disclosure uses a species-specific test for companion animals suchdogs or cats, and other animal species, and the appropriate methods.

This disclosure relates to a diagnostic test system for screening ortesting for sensitivity or intolerance to pet food constituents orcompositions for a domestic animal, particularly for a dog or cat, andfor other species, such as food and fiber animals, and horses.

In particular, the disclosure relates to a diagnostic test system forscreening or testing for sensitivity or intolerance of a complete foodor food supplements in a dry form, semi-dry form, powdered, or a wetform integrated with functional or nutraceutical compounds from plant orother origins.

The disclosure relates to a diagnostic test system for screening ortesting for sensitivity or intolerance to different botanicals or othermicronutrients. The activity and the efficacy of the botanicals or othermicronutrients depend upon the individual genetic make up of anindividual.

The disclosure uses genetic information of DNA polymorphism, thefunctional genomic profile, and the different response of an individualanimal to a biologically active nutrient in order to identify andimprove upon or optimize the nutrient composition of the diet for anindividual animal.

A unique feature of the disclosure is that the response to abiologically active nutrient ingestion or exposure is a dynamic eventsince it depends upon the genetic variants of nutritionally induciblegenes (polymorphisms, as SNPs) that can lead to a different effect ofthe biologically active nutrient in individual animals having differentgenotypes.

Effectively, the genotype of the individual animal is an essentialcomponent of this disclosure to permit the identification of thebiologically active nutrient for that individual animal.

The assessment of the biologically active nutrient composition of thediet arises from using reference data relating to healthy animals withdifferent genotypes, plus target data relating to animals affected withdifferent physiological or pathophysiological states [termed “unhealthyanimals”] and having different genotypes, and nutritional data relatingto the different effects of nutritional compounds in healthy andunhealthy animals or groups of animals with different genotypes.

Additional and further objects, features, and advantages of the presentdisclosure will be readily apparent to those skilled in the art.

DRAWING

FIG. 1 is a schematic illustrating the determining and prescribing of aPhase I special cleansing diet food regime in conjunction with the foodsensitivity and intolerance testing. The testing is conducted in aveterinary laboratory or by Point of Service (“P-O-S”) device. The dietshere are for liver/GI tract, antiarthritic, anti-inflammatory,anti-anti-allergy and vegetarian. There may be less or more of thesediets. The testing is interpreted by machine, reader or computer andthen reported electronically.

FIG. 2 is a schematic illustrating the determining and prescribing of aPhase II “Other Special Needs Diets” food regime in conjunction with thefood sensitivity and intolerance testing. The testing is conducted in aveterinary laboratory or by P-O-S device. The diets here are forgeriatric, obesity, immune enhancement, sport or endurance purposes.There may be less or more of these diets. The testing is interpreted bymachine, reader or computer and then reported electronically.

DETAILED DESCRIPTION OF THE DISCLOSURE

There is a need to facilitate the choice of a pet food to suit selectedanimals so that there is compatibility between the pre-prepared balancedand integrated food composition or mixture of macro- andmicro-ingredients and the physiological, metabolic, biochemical orgenetic makeup of the pet or companion animal. Most companion animalsare fed commercially pre-mixed, pre-compounded, or constituted macro-and micro-ingredients such that there is a single integrated food withsuch pre-mixed and pre-integrated ingredients. This is usually stated tobe the complete or main stay or basic food or diet for the companionanimal. Thus, normally such companion animals receive as their regularso-called healthy and nutritious diet one or more regular pre-packagedmixtures of ingredients in commercially prepared foods.

This is largely different to human diets which are generally notpre-packaged as a complete single food with the necessary macro- andmicro-ingredients for a complete and healthy diet. Instead the humandiet is typically formed with variety and is selected from multipleingredients which are assembled by choice or purpose, and specificallyprepared and cooked on a custom basis according to the individual choiceand preference of a human, and specifically for different meals or mealcombinations.

The food ingredient or ingredients for which the method of thedisclosure is performed is contained in a composition being at least oneof a pre-processed food composition, balanced diet or recipecomposition. The testing is performed to identify which ingredient(s)from that group of pre-packaged and pre-mixed companion animal foodscreate sensitivity or intolerance. This can be a moist or semi-moistfood, dry kibble or an extruded cereal product. The majority ofcompanion animals or pets in developed countries are fed by thiscommercially generally available off the shelf pre-prepared pet foodmixtures. Sensitivities or intolerances to these foods often arise, butthere is no simple manner of determining which ingredient(s) in thosefoods causes the sensitivity or intolerance.

The disclosure relates to multistage or staged testing for foodsensitivity from different test protocols. There is a saliva based testprotocol, followed by a serum based test protocol and then a DNA/RNAnutrigenomic test protocol. In other cases there is staged testing froma first test protocol, being a serum based test protocol and then aDNA/RNA nutrigenomic test protocol. There is also a system for providingstaged testing from a first test protocol, being a saliva based test,followed by a DNA/RNA nutrigenomic test protocol.

In many cases there are a high proportion of animals requiring thesaliva testing, and the testing is performed frequently during a lifespan of an animal. Such testing can often be performed by a lay personsuch as the owner of the animal. The serum stage requires the taking ofblood from the animal, usually by a veterinarian or veterinarytechnician. This testing will be required less frequently during thelifetime of the animal. The most sophisticated testing, the nutrigenomicheat map testing, will be required less frequently by a smallerpercentage of the overall population of animals. This required a bloodsample from the animal and processing in a high throughput (“HTP”)DNA/RNA measuring system in a laboratory.

The present disclosure permits for integrated single stage testing stepsfor multiple antigens following the intake of a pet or companion animalmulti-ingredient food product. The single stage bodily fluid test, testsfor multiple antigens in the food product.

In one form of the disclosure, the companion animal is tested by givingsuch a food being a pre-prepared integrated pre-mixture of ingredientscontaining the multiple macro- and micro-ingredients for the animal.Then the animal is subjected to at least one of the testing anddiagnostic procedures. In this manner it is possible to determine whichingredient(s) in a pre-processed food composition, balanced diet orrecipe induces a possible sensitivity or intolerance issue. Anassessment is made as to whether different pre-processed or pre-packagedfood compositions, balanced diets or recipes are available to offer moreappropriate, compatible food(s). In some cases it may necessary todetermine whether a special diet needs to be formulated without theoffending ingredient or ingredients.

Food sensitivity or intolerance has an immunological basis. It is notpossible to distinguish a food which elicits an immunological responsefrom the related intestinal disease or disorder that reflects the body'sreaction to the food ingredient or ingredients. The description providedis primarily related to detecting immunologic food sensitivities orintolerances in animals.

A primary example of a food sensitivity or intolerance is sensitivity towheat or other gluten foods, for example barley, rice, millet, and oats.In the Irish Setter breed, for example, wheat-sensitive enteropathy is aheritable condition. Immunological reactions to gluten foods causesatrophy of the intestinal villi and inflammation of the small intestine,which, in turn, results in diarrhea and weight loss from malabsorptionof fluid, electrolytes, and dietary nutrients. Even though chronic orintermittent diarrhea and intermittent vomiting are the most commonsymptoms of this food sensitivity, there have been few studies of theprevalence of this condition in animals being presented to veterinarianswith chronic diarrhea or vomiting or other common gastrointestinalsymptoms. Furthermore, beyond costly measurements of serum IgE-mediatedantibodies, there are no adequate methods in veterinary medicine todiagnose or noninvasively test for immunologic food sensitivities orintolerance. This frequently results in either no diagnosis or themissed diagnosis of an immunologic food sensitivity or intolerance.

Despite this situation, many animals with gluten or other foodsensitivity or intolerance do not have diarrhea or weight loss, butinstead have other signs and symptoms such as vague abdominal pain,nausea, abdominal bloating, flatulence, chronic fatigue, constipation,poor growth and maturity, iron deficiency anemia, osteoporosis, seizuresor other neurologic disorders, or even just elevated serum liver enzymelevels. Some animals may be asymptomatic.

Furthermore, animals with gluten or other food sensitivity orintolerance may not have fully developed intestinal lesions. Therefore,the food sensitivity or intolerance of these animals may not be properlydiagnosed using known testing methods, such as endoscopic intestinalbiopsy and blood or serum testing. Additionally, these animals maypresent with other immunologic diseases such as the autoimmune diseasesof skin, liver, joints, kidneys, pancreas, and thyroid gland, ormicroscopic colitis.

One form of the disclosure relates to a method for diagnosing a foodsensitivity or intolerance in a companion animal comprising the steps offirstly collecting a saliva sample; screening the saliva or blood spotsample to detect the qualitative or semi-quantitative or quantitativepresence of at least one of an IgA, IgM or IgG antibody to a particularfood ingredient or composition. Diagnosing a food sensitivity orintolerance based on the presence of the antibody, is then effected.

A second form of the disclosure involves collecting a blood sample; andpreparing serum from the sample; screening the serum sample to detectthe qualitative or semi-quantitative or quantitative presence of atleast one of an IgA, IgM or IgG antibody or immune complex to aparticular food ingredient or composition. Diagnosing a food sensitivityor intolerance based on the presence of the antibody or immune complexis affected.

In some cases the first and second steps are selectively divided intotwo stages, the first stage being a qualitative step to determine thefood sensitivity, followed by a semi-quantitative or quantitative step.

This disclosure is based on an Enzyme-Linked Immunosorbant Assay(ELISA), or other immunoassay platforms such as but not limited tolateral flow, or latex or bead agglutination, which measures thepresence of at least one of IgA, IgM or IgG antibodies or immunecomplexes against a wide variety of foods or food supplements or foodadditives in an animal's serum, as well as at least one of IgA, IgM orIgG antibodies in an animal's saliva or blood spot or other bodilymatter.

The current disclosure measures at least one of, and preferably morethan one of serum IgA, IgM or IgG or immune complexes that may bemediated by complement. Also, at least one of, and preferably more thanone of salivary or blood spot or other bodily fluid IgA, IgM or IgG ismeasured.

The amount of these antibodies in serum and saliva or blood spot orother bodily matter of healthy individual animals is compared to that inserum and saliva or blood spot or other bodily matter of animals withclinically expressed or suspected pre-clinical or latent foodsensitivity or intolerance.

One method associated with the disclosure is for diagnosing a foodsensitivity or intolerance in companion animals which comprises thesteps of: collecting a blood sample; preparing serum from the sample;screening the serum sample to detect the presence of at least one of,and preferably more than one of an IgA or IgG antibody or immune complexto a particular food ingredient or composition. Thereafter there is adiagnosis of a food sensitivity or intolerance based on the presence ofthe antibody or immune complex. Another method associated with thedisclosure is for diagnosing an food sensitivity or intolerance incompanion animals comprising the steps of: collecting a saliva or bloodspot sample; screening the saliva or blood spot sample to detect thepresence of at least one of, and preferably more than one of an IgA orIgM or IgG antibody or immune complex or immune complex with complementto a particular food ingredient or composition. Thereafter there is adiagnosis of a food sensitivity or intolerance based on the presence ofthe antibody or immune complex or immune complex with complement.

The method for diagnosing a food sensitivity or intolerance in acompanion animal can comprise the steps of obtaining a sample of aserum, non-serum biological fluid, selectively saliva or blood spot orother non-serum bodily fluid or matter. The sample is screened to detectthe presence of at least one of an IgA, IgM or IgG antibody or immunecomplex or immune complex with complement to a particular foodingredient or composition. A food sensitivity or intolerance isdiagnosed based on the presence of the antibody or immune complex orimmune complex with complement.

Where the sample is saliva or blood spot or other bodily fluid ormatter, a testing portion from the saliva or blood spot or other bodilyfluid or matter is collected and the testing portion is the sample inthe screening step. The sample is about 1-5 milliliters or about 1-15grams.

The screening step utilizes an immunoassay, selectively an enzyme-linkedimmunosorbant assay (ELISA) testing system to detect the presence of anantibody immune complex or immune complex with complement to aparticular food ingredient or composition of food ingredients.

Another method associated with the disclosure is for diagnosing a foodsensitivity or intolerance in companion animals comprising the steps ofcollecting a saliva or blood spot sample; screening the saliva or bloodspot sample to detect the presence of at least one of, and preferablymore than one of an IgA or IgM or IgG antibody to a particular foodingredient or composition.

Thereafter there is a diagnosis of a food sensitivity or intolerancebased on the presence of the antibody, and collecting a blood sample;preparing serum from the sample; screening the serum sample to detectthe presence of at least one of, and preferably more than one of an IgA,IgM or IgG antibody or immune complex to a particular food ingredient orcomposition.

Thereafter there is a diagnosis of a food sensitivity or intolerancebased on the presence of the antibody or immune complex.

The food sensitivity is at least one of wheat or other glutensensitivity or intolerance, corn or soy, beef or other meat or fishprotein sensitivity or intolerance or dairy, eggs, other grains,botanicals, oils from seeds or fish, vegetables, or fruit sensitivity orintolerance.

The food ingredient for which sensitivity or intolerance is being testedis contained in at least one mixed ingredient food having multipleingredients in varying amounts of a premixed food composition, balanceddiet or recipe.

The companion animal is tested by giving such a mixed food to theanimal. The animal is then tested and a sensitivity or intolerancediagnosis is made by determining the reaction of the animal to differentfoods and specific selected ingredients. In this manner it is possibleto determine whether there is and if so which ingredient in apreprocessed food composition, balanced diet or recipe is a potential orreal sensitivity or intolerance problem for a specifically testedanimal. An assessment is made as to whether it is possible to use adifferent preprocessed mixed food composition, balanced diet or recipe,or whether a special diet needs to be formulated without the offendingingredient or ingredients.

The test methodology of the present disclosure differs significantlyfrom all others available for use in animals. It is highly reproducibleand clinically relevant. In serum, the food antigen or peptide beingtested and at least one of, and preferably more than one of any specificIgA or IgG antibody in serum, bind to each other and then fixcomplement. In saliva or blood spot or other bodily fluid, the foodantigen or peptide being tested reacts directly with the IgA or IgMantibody in the test animal's saliva or blood spot or other bodilyfluid. Common animal food antigens (such as wheat or other glutens, cornand soy, beef or other meats, fish or other foods and botanicals) arebound to wells in a 96-well standard ELISA microtiter plate so that theyare non-reactive until an animal's serum or saliva or other bodily fluidis added. Specific binding of IgA or IgG antibody or immune complexes inserum or IgA or IgM antibody in saliva or blood spot or other bodilyfluid to specific food ingredients is identified by finding increasedlevels of one or more of these antibodies in test, unhealthy or suspectindividuals in comparison to healthy control animals.

In another embodiment, the salivary or blood spot or other bodily fluidtesting uses a quantitative or semi-quantitative point-of-service(P-O-S) test kit system, whereby the animal owner or veterinarian orother designated tester collects the saliva or other bodily fluid fromindividual healthy animals or unhealthy or suspect animal patients. Therequisite biological sampling swabs or straws are provided in the P-O-Stest kit; the collected saliva or other bodily fluid is added to thetest kit chamber, and the chamber is sealed and submitted to the testinglaboratory for quantitative analysis. This is a single stage test formultiple antigens.

In another embodiment, the salivary or other bodily fluid testing uses aquantitative point-of-service (P-O-S) test kit system, in which therequisite salivary or other bodily fluid biological sampling swabs orabsorbent paper or straws are provided. The collected saliva or otherbodily fluid is added to a special well in the test kit chamber, whichis then allowed to react by capillary attraction with a series ofmicrochannels containing various food antigens or peptides of interestwithin the chamber's lateral flow device. Once the reaction iscompleted, the chamber is sealed and submitted to the testing laboratoryfor semi quantitative or quantitative analysis. This is a single stagetest for multiple antigens.

In a further embodiment, the salivary or blood spot or other bodilyfluid testing uses a quantitative r semi-quantitative point-of-service(P-O-S) test kit system, in which the requisite salivary or blood spotor other bodily fluid biological sampling swabs or absorbent paper orstraws are provided. The collected saliva or blood spot or other bodilyfluid is added to a special well in the test kit chamber or dipped intoa test strip, which is then allowed to react by capillary attractionacross a reagent strip containing various food antigens or peptides ofinterest within the strip, much like the existing urine dip-sticktechnology strips, routinely used in human and veterinary medicine. Oncethe reaction is completed, qualitative reactions are read on acolor-grading scale provided with the kit. These reactions can rangefrom negative or little to no color reaction to a highly reactiveintense color development. This is a single stage test for multipleantigens.

Positive qualitative reactions seen with the P-O-S test kit can help toidentify the major food reacting antigens or peptides in an animal'ssaliva or blood spot or other bodily fluid, but should be confirmed byone or more of the semi quantitative or quantitative serum or saliva orblood spot or other bodily fluid tests described in this disclosure.This is a single stage test for multiple antigens.

For semi-quantitative or quantitative testing, an animal's serum orblood spot or saliva or other bodily fluid is added to the ELISAmicrotiter plate or other immunoassay platforms such as but not limitedto lateral flow, or latex or bead agglutination, which measures thepresence of IgA, IgM or IgG or immune complexes against a wide varietyof foods or food supplements or food additives in an animal's serum, aswell as in an animal's saliva or blood spot or other bodily fluid andany specific antibodies present directed against IgA, IgM or IgGantibodies or immune complexes (serum) or IgA, IgM or IgM antibodies(saliva) or blood spot or other bodily fluid are then bound to theirrespective food antigens or peptides. The plate is washed and an enzymeconjugate is added that recognizes the bound antibodies of IgA, IgM orIgG or immune complexes in serum, and IgA or IgM in saliva or blood spotor other bodily fluid. After incubation and washing, substrate is addedto visualize the bound antibodies of IgA, IgM or IgG or immune complexesin serum, and IgA or IgM or IgG in saliva or blood spot or other bodilyfluid. The amount of the optical density recorded is proportional to theamount of bound antibody to IgA, IgM or IgG or immune complexes inserum, and IgA or IgM or IgG in saliva or other bodily fluid. A reportdepicting these reactions is plotted on a simple bar graph or by othermeans. Results show with a high degree of accuracy if an animal patienthas a positive or negative reaction against a particular foodingredient.

Food Sensitivity or Intolerance Reactions

Research to date has shown that serum IgA or IgG alone, or withcomplement, constitute the main immunologically reactive pathways offoods and food supplements. In saliva or other mucosal secretions orother bodily fluids such as tears or milk, the main immunologicreactants are IgA or IgM. These reactive immune responses arecharacterized classically as Types I, II, III, and IVhypersensitivities.

Type I immune reactive responses are mediated by IgE antibody, and arecommonly called an immediate hypersensitivity. This allergic reactionoccurs within two hours of allergen exposure or ingestion.

Type II immune reactive responses are mediated by IgG or IgM antibodiesand are commonly called delayed hypersensitivity. The allergic reactionoccurs from two hours to several days after allergen exposure.

Type III hypersensitivity responses form an immune complex that is alsoa delayed hypersensitivity, because the allergic reaction occurs days toweeks post allergen exposure or ingestion. Type III reactions developwhen immune complexes, typically of the IgG class form in such largequantities that they cannot be cleared adequately by thereticuloendothelial system. Allergen exposure results in production ofIgG, which then binds to the allergen, forming immune complexes in theblood. These immune complexes in turn activate complement, resulting inthe covalent binding of complement component C3b to the IgG, therebyforming immune complex-C3b. The immune complexes so formed are depositedat various sites throughout the body, and damage ensues when they aredeposited and further activate complement, producing and releasinginflammatory cytokines. Release of cytokines causes leukocytes torelease proteases, mast cells and vasoactive amines that damage bloodvessels and escalate the inflammatory process.

Type IV immune reactive responses represent the cell-mediated form ofdelayed hypersensitivity. The allergic reaction occurs days to weeksafter allergen exposure. The most serious type of delayedhypersensitivity is a granulomatous tissue rejection, which occurs whenmacrophages ingest but cannot degrade an offending allergen, therebyresulting in persistent stimulation of tissue macrophages. Stimulatedmacrophages elaborate cytokines that cause the macrophages and othercell types to concentrate around and in the area of tissue injury.T-lymphocytes are then stimulated in turn by cytokines, which activatecomplement and induce immune-complex formation.

Measuring IgE remains the standard for determining the presence ofinhalant allergies (atopy), but this method usually fails or is toocostly when diagnosing food sensitivity or intolerance as related tochronic diseases.

Delayed food-related sensitivities begin in the gastro-intestinal tractwhen the intestinal lining becomes hyperpermeable. This problem is knownas “leaky gut syndrome” or intestinal dysbiosis, and is defined as anincrease in permeability of the intestinal mucosa to partially digestedprotein macromolecules, micromolecules, antigens and toxins. Theimmunological reaction to these proteins or other molecules in the liverinitiates and perpetuates chronic food sensitivity or intolerance. Whenthe gut is unhealthy, the rest of the body is unhealthy. The diseaseprocess that ensues is typically chronic or intermittent and ofteninvolves the gut and skin, as well as internal organs such as the liver.Gastro-intestinal tract function is disrupted when the lining of the gutis inflamed or damaged. With a leaky gut, large food antigens can beabsorbed into the body. The body's defense systems then attack thisantigen or antigens and the result is the production of antibodiesagainst what was once a harmless, innocuous food ingredient. These IgAor IgG antibodies and immune complexes are formed in the bloodstream andcirculate throughout the body where they can damage other tissues alongthe way. In saliva or other bodily fluid, these reactants are typicallyIgA or IgM.

Immune Complex

Immune complexes containing large food antigens enter the blood from thegastro-intestinal tract then travel through the liver where most immunecomplexes are removed. However, if circulating immune complexes pass theliver filtering system, they may cause injury to many body tissues.Malabsorption of food particles from the gastro-intestinal tract canalso travel by lymphatic drainage to the body. The lymph channels in thegut wall converge at the thoracic duct which drains its contents intothe large thoracic veins. This combination of antibody with complementin the blood stream becomes a circulating immune complex. Immunecomplexes subsequently attach to receptors on red and white blood cellsand then these altered cells are cleared by the body's liver or spleen(reticuloendothelial system).

Any circulating immune complexes that are not removed by thereticuloendothelian system of the liver (or spleen) can activate thecomplement cascade. Individuals with more immune complexes on their redblood cells are the ones that suffer from chronic food sensitivities orintolerances.

Circulating immune complexes also can damage the integrity of bloodvessel capillaries which in turn can trigger inflammatory events.

Diagnosing Immunologic Food Sensitivity or Intolerance by Blood orSaliva Testing Samples for Antibodies or Immune Complexes

A blood serum or saliva or blood spot or other bodily fluid sample fromthe animal patient is shipped to the laboratory for testing. Althoughthis is one method for collecting a sample from a patient, it isrecognized that other methods of obtaining a sample may be used withinthe scope of the disclosure. Such methods include taking the patientphysically to a veterinary clinic or laboratory to collect a bloodsample to prepare its serum, or a saliva or blood spot or other bodilyfluid sample, or, could include a P-O-S qualitative saliva or blood spotor other bodily fluid screening test performed by the pet owner orveterinarian or other person.

Once collected at or received by the lab, the blood serum or blood spotor saliva or other bodily fluid sample is then screened using the ELISAmethod or other immunoassay platforms such as but not limited to lateralflow, or latex or bead agglutination, which measures the presence ofselected IgA or IgG antibodies or immune complexes against a widevariety of foods or food supplements or food additives in an animal'sserum, as well as IgA or IgM antibodies in an animal's saliva or bloodspot or other bodily fluid. The detection of a particular antibody inthe patient serum or saliva or blood spot or other bodily fluid at alevel higher than that seen in healthy animal patients then forms thebasis for a diagnosis of the food sensitivity or intolerance associatedwith that antibody.

In one form there is provided a diagnostic test system for screening ortesting for food sensitivity or intolerance in animals of the foodcomposition which can comprise at least several active components ofwheat or other gluten foods, corn, soy, beef or but not limited to othermeats, fish, dairy, eggs, other grains, botanicals, oils from seeds orfish, botanicals, vegetables, nuts, or fruit sensitivity or intolerance.

In another form there is provided a diagnostic test system for screeningor testing for food sensitivity or intolerance in animals of otherconstituents including a group of and at least macro- andmicro-components such as vitamins, amino acids, and one or more plant,part of plants and plant extract(s) having functional and nutraceuticalproperties.

There is also provided a diagnostic test system for screening or testingfor food sensitivity or intolerance in animals of the activity of one ormore protein compounds, depending upon the food ingredients, thephysiology of the animal's digestive tracts and the individual geneticmake up of the animal.

There is provided a diagnostic test system for screening or testing forfood sensitivity or intolerance in animals that individually respondwith an immune reactive response to functional, nutraceutical ortherapeutic compounds, and which depends upon the genetic make up of theanimal, which differs from the ability to adapt to the environment andto interact with nutrients.

There is provided a diagnostic test system for screening or testing forfood sensitivity or intolerance in animals that the activity and theefficacy of functional, nutraceutical and therapeutic compounds of plantor other origin depends upon their molecular dietary signature, which isrelated to the individual genetic make up, according to the concept ofnutrigenomics.

The immune reactive response of a pet animal is dependent upon itsgenetic make up that is the coordinated and integrated relationshipamong genes which can be differently regulated from botanicals or othercompounds, according to the individual genotype; the gene(s) actdownwards (down-regulated) and upwards (up-regulated) in the regulationof molecular, cellular and biological pathways.

There is provided a diagnostic test system for screening or testing forfood sensitivity or intolerance in animals that wherein the adverseimmunologic reaction effect of the ingredients can also vary accordingto the genotype of pet animals, and the variability of individualanimals.

The compounds and food ingredients can induce a variable reactive immuneresponse to the physiological or pathological response of an individualpet animal. This can be expressed by what is termed “leaky gut syndrome”or intestinal dysbiosis. Furthermore, the gut dysbiosis in turn can leadto damage or malfunction of other body tissues, especially the skin.This is termed the “gut-skin connection.”

The term “pet” means a domestic dog, cat or horse. The term “pet foodcomposition” means a food composition or feed ingredient that isintended for ingestion by the pet. Pet food compositions may include,without limitation, nutritionally balanced compositions suitable fordaily feed, as well as supplements which may or may not be nutritionallybalanced.

The pet food compositions that induce adverse immune reactions or foodsensitivities may be prepared by any of a variety of processes. Thecomponents may be obtained from plant or vertebrate animal matter, orotherwise provided, and then subjected to pet food processing, as meal,pellet, cold extrusion, heat extrusion autoclaved tins and pouches.

There is provided a diagnostic test system for screening or testing forfood sensitivity or intolerance in animals, of the offending foodingredients being in preprocessed or premixed pet food meals, biscuits,snacks, treats, sprinkles, candies and other form of foods.

The ELISA testing system, or other immunoassay platforms such as but notlimited to lateral flow, or latex or bead agglutination, are well knownin the art. These assays measure the presence of selected IgA or IgGantibodies or immune complexes against a wide variety of foods or foodsupplements or food additives in an animal's serum, as well as IgA orIgM antibodies in an animal's saliva or blood spot or other bodilyfluid. The presence of these IgA or IgG antibodies or immune complexesin a patient's serum is tested against one or more of a selected panelor group of food antigens or ingredients. The presence of IgA or IgMantibodies in saliva or blood spot or other bodily fluid is also testedagainst one or more of a selected panel or group of food antigens oringredients. If the results show an increased level of any of theseselected antibodies to food antigens or ingredients in the serum orsaliva or blood spot or other bodily fluid of a patient in comparison tothose levels of the same selected antibodies in healthy animals, thefindings indicate that the animal patient does in fact have a particularimmunologic food sensitivity or sensitivities or intolerance. The animalpatient should then be treated accordingly by removing the triggeringsubstance or substances from the diet or by other methods known in theart.

Although specific parameters and equipment have been discussed in thisaspect of the disclosure, it is understood that the parameters maydiffer and that different equipment may be used to carry out thedisclosed methodologies without deviating from the scope of thedisclosure.

This blood serum or saliva or blood spot or other bodily fluid testingmethod may be combined with one or more other immunologic foodsensitivity or intolerance diagnostic indicators previously describedaccording to other established methods in order to further enhance thesensitivity and accuracy of immunologic food sensitivity or intolerancediagnosis.

Secretory IgA serves as a mucosal barrier to certain macromolecules,bacteria, and viruses. When these molecules or organisms interact withsecretory IgA and the mucosa, their entrance and exposure to thegut-associated lymphoid tissue (GALT) is blocked. This blockage permitsthe host to shield efficiently the systemic immune response, localimmune response, or both, from onslaught of foreign molecules.

Secretory IgA therefore has anti-bacterial, anti-fungal, and anti-viralactivities, and plays an important role in protection of mucosalsurfaces from adherence of microorganisms.

Another important role of secretory IgA is in prevention of thediffusion of food antigens into mucous membranes.

Despite the enteric route of exposure to food antigens and peptides,food-specific antibodies are typically measured only in blood, and notin saliva or other bodily fluid.

The disclosure includes using a bodily fluid of a companion animal suchas saliva to determine food sensitivity of that animal. Saliva is anaccessible fluid, easy to collect, and demonstrates representativeresponses in secretions after enteric or intragastric antigenicchallenge.

The disclosure includes measuring at least one of, and preferably morethan one of salivary or other bodily fluid IgA or IgM against differentfood antigens and peptides for use in determining food allergy and foodintolerance in companion animals.

A method for determining the presence of food allergy or foodintolerance in a companion animal such as a dog or cat or horse includes(a) determining at least one of, and preferably more than one of thelevel of salivary or other mucosal or other bodily fluid IgA or IgMantibody or antibodies against a dietary antigen or peptide present in afood in a saliva or other mucosal or other bodily fluid sample from theanimal; and (b) comparing the level determined in step (a) with normallevels of the antibody or antibodies in the mucosal or other bodilyfluid sample.

The possible outcomes for the comparison include (i) lower than normallevels or about normal levels of dietary antigen antibodies indicateoptimal conditions; and (ii) higher than normal levels of dietaryantigen antibodies indicate a food allergy or food intolerance.

There is a also a method for determining a type of antibody in apresence of food allergy or food intolerance to a food in an animal,comprising (a) determining a level of antibodies against a dietaryantigen or peptide present in the food in a first saliva or othermucosal or other bodily fluid sample from the animal patient; (b)determining a level of antibody or antibodies against cross-reactivetissue antigen or antigens or peptides present in a second salivary orother mucosal or other bodily fluid sample from the animal patient,wherein the first and second samples are the same or different; and (c)comparing the level of antibody or antibodies determined in steps a) andb) with normal levels of the antibody or antibodies found in healthyanimals.

The possible outcomes for the comparison include (i) essentially normallevels of antibody or antibodies against the dietary antigen or peptideand normal levels of antibody or antibodies against cross-reactivetissue antigen or peptide indicate optimal conditions; (ii) higher thannormal levels of antibody or antibodies against the dietary antigen andessentially normal levels of antibody or antibodies againstcross-reactive tissue antigen or peptide indicate food allergy andintolerance without cross-reacting to tissue antigen or peptide; (iii)essentially normal levels of antibody or antibodies against the dietaryantigen or peptide and higher than normal levels of antibody orantibodies against cross-reactive tissue antigen or peptide indicate anautoimmune reaction not related to the dietary antigen or peptide; and(iv) higher than normal levels of antibody or antibodies against thedietary antigen or peptide and higher than normal levels of antibody orantibodies against cross-reactive tissue antigen or peptide indicate apresence of food allergy and intolerance resulting in an autoimmunereaction.

A test informs a pet owner of clinical conditions of their pet who maysuffer from food sensitivities, allergies or food intolerance. The testuses a method that measures antibody titers to dietary antigens. Thetest method measures the antibodies' ability to bind to a recombinantantigen, synthetic peptide, a peptide prepared by enzymatic digestioncorresponding to the dietary antigen, or different cross-reactive tissueantigen or antigens.

There is an immunoassay for detecting food allergies and foodintolerance in a patient using mucosal or other bodily fluid secretions.Mucosal secretions are secretions of a mucosa, such as saliva.

Forms of biological fluid, other than saliva, for instance, blood,feces, urine, tears, or milk or other mucosal secretions can be used.

There is a method for detecting food allergies and food intolerance inan animal. The method includes (a) determining a level of antibody orantibodies against a dietary antigen or peptide in the food in asalivary or other mucosal or other bodily fluid sample from a patient;and (b) comparing the level determined in step (a) with normal levels ofthe antibody or antibodies in the salivary or other mucosal or otherbodily fluid sample.

The possible outcomes for the comparison include (i) lower than normalor essentially normal levels of antibody or antibodies to dietaryantigen or peptide indicate optimal conditions; and (ii) higher thannormal levels of antibody or antibodies to dietary antigen or peptideindicate a food allergy or food intolerance.

The detection of antibodies can be performed with an immunoassay.Immunoassays include, but are not limited to, ELISA test, RIA test,latex agglutination, beads assay, and proteomic assays. A preferableimmunoassay is the ELISA test. Other immunoassays can be used and thechoice of immunoassay can be determined by one of ordinary skill in theart.

A normal reading is derived from a baseline measurement taken fromantibody measurements for individuals without symptoms relating to foodallergies or food intolerance. A baseline measurement for the test isobtained by observing the antibody measurements for individuals withoutsymptoms relating to food allergies or food intolerance. For example,most readings for antibody measurements from an individual withoutsymptoms relating to food allergies or food intolerance are below acertain reading. Preferably, about 50-100% of the readings from ananimal without symptoms relating to food allergies or food intoleranceare below the certain reading, more preferably about 60-100%, 70-100%,or 80-100% of the readings, even more preferably about 90-100% of thereadings. If an animal exhibits antibody measurement two standarddeviations above the baseline, the above-normal antibody measurementindicates the presence of food allergy or food intolerance.

Additionally, antibodies against cross-reactive tissue antigen orantigens may be tested. Cross-reactive tissue antigen or antigensinclude, but are not limited to, lectins, lectins receptors,tropomyosin, smooth muscle, epithelial cell antigens, enzymes,cytochrome P-450 enzymes, and transglutaminase. Ingested dietaryantigens or peptides may induce antibodies that react with the specificdietary antigen and another antigen, such as a cross-reactive tissueantigen. If antibodies against cross-reactive tissue antigens are testedin addition to the dietary antigens, then the antibodies can bedetermined to be protective or pathogenic.

Essentially normal levels of antibodies against the dietary antigen orpeptide and normal levels of antibodies against cross-reactive tissueantigen or antigens indicate optimal conditions. Higher than normallevels of antibodies against the dietary antigen or peptide andessentially normal levels of antibodies against cross-reactive tissueantigen or antigens indicate food allergy and intolerance withoutcross-reacting to tissue antigen or antigens. Essentially normal levelsof antibodies against the dietary antigen or peptide and higher thannormal levels of antibody or antibodies against cross-reactive tissueantigen or antigens indicate an autoimmune reaction not related to thedietary antigen or antigens. Higher than normal levels of antibodiesagainst the dietary antigen or peptide and higher than normal levels ofantibodies against cross-reactive tissue antigen or antigens indicate apresence of food allergy and intolerance resulting in an autoimmunereaction.

There can be an apparatus and method for conducting a variety of assaysfor the determination of analytes in samples. There can be a single-usedevice designed to be adaptable to a variety of real-time assayprotocols, preferably assays for the determination of analytes inbiological samples using immunosensors or other ligand/ligandreceptor-based biosensor embodiments.

There can be a metered portion of a sample, for precise and flexiblecontrol of the movement of a sample or second fluid within the device.The device and method is for rapid in situ determinations of one or moreanalytes, and single-use methodology that minimizes the risk ofcontamination of both operator and the animal. As such there is apoint-of-service (P-O-S) clinical diagnostic use.

A multitude of laboratory tests for analytes of interest are performedon biological samples for diagnosis, screening, disease staging,forensic analysis, pregnancy testing, drug testing, and other reasons.While a few qualitative tests, such as pregnancy tests, have beenreduced to simple kits for home use, the majority of quantitative testsstill require the expertise of trained technicians in a laboratorysetting using sophisticated instruments.

Optical means for detecting the binding of an analyte to a receptor isemployed, or alternatively there can be electrochemical detection, inwhich binding of an analyte directly or indirectly causes a change inthe activity of an electroactive species adjacent to an electrode, hasalso been applied to immunoassay.

Therefore, there exists within the field of analyte sensing, and inparticular for applications in which analytes must be determined withinbiological samples such as blood, a need for apparatus that can rapidlyand simply determine analytes at the P-O-S, and can be performed by lesshighly trained staff than is possible for conventional laboratory-basedtesting. It would be of benefit in the diagnosis and treatment ofcritical veterinary conditions for the veterinarian or veterinarytechnician to be able to obtain clinical test results without delay. Theapparatus should be adaptable to determination of a range of analytesand capable of single-use so that there can be disposal of the sampleafter testing.

A device according to the present disclosure has the advantage that thesample and a second fluid can contact the sensor array at differenttimes during an assay sequence. The sample and second fluid may also beindependently formed with other reagents or compounds present initiallyas dry coatings within the respective conduits. Controlled motion of theliquids within the device further permits more than one substance to beadded into each liquid whenever the sample or fluid is moved to a newregion of the conduit.

In operation, an amount of a preferably biological sample is placed intothe sample chamber of the device. The device can have reading zones orthe device can be placed into a reading apparatus. A metered portion ofthe sample can be amended with at least one antibody-enzyme conjugate,and is then contacted with the immunosensor. A second fluid, whichcontains an inactive substrate for the enzyme, is used to rinse theimmunosensor substantially free of unbound antibody-enzyme conjugate,and the response of the immunosensor is recorded and analyzed for thepresence, or amount of, analyte of interest. The device may contain aplurality of immunosensors and reagents.

After the reading, the operator removes and discards the device. Thereader is then ready for another measurement. While the use of thedisclosure is frequently referred to in a biological or medical context,it will be appreciated that the present disclosure may be practiced inany situation where it is desired to perform in situ chemical analysesof liquid samples at speeds which approach real-time.

A dipstick test device is used for detecting an analyte in a liquidsample such as saliva or other biological fluid by treating the analytewith at least one liquid reagent to form a detectable reaction product.The device can include: a) an aqueous impermeable, aqueous insolublereaction zone, adapted to retain the detectable reaction product; and b)a control absorbent above, and in liquid-transferring relation with, thereaction zone. The control absorbent can have predetermined, limitedliquid-absorbing capacity, and the dipstick is configured for locationwith a vessel containing the sample. The control absorbent is above thereaction zone, so that the control absorbent fills with sample and thereaction zone incubates with the sample. The device may further includean absorbent reservoir which can move into liquid transferring contactwith the reaction zone.

This device and method is for use for detecting an analyte, for example,using an immunoassay. An analyte in a sample may be detected by treatingthe sample with various reagents, such as labeled immunological bindingpartners to the analyte and reagents to enable detection of the label.Often, the sample is washed between administrations of various reagents.

An assay may depend on controlling the amount of reactants exposed tothe sample and the duration of the reactions taking place. It isdesirable to have the ability to assay small sample volumes withrelative low concentrations of analyte, and/or to detect relativelysmall differentials in analyte concentration. Finally, it is desirableto have a system to permit measuring whole blood samples, serum andsaliva or other bodily fluid without complex equipment.

One method for adding and washing reagents in an immunoassay uses anabsorbent material to move liquid washes and reagents through a solidsubstrate such as a membrane to which other reactants are immobilized.

There can be an immunoassay test device including sorbent material fordrawing liquid through a microporous membrane at the bottom of a testwell. The sorbent material is resiliently biased away from the membrane,and it draws liquid through the membrane only when the two are forcedtogether to overcome the bias. Sorbent material comprises a surfacelayer which is hydrophobic and a bulk portion which is wettable.Reagents are added serially to the test well and, after each reagent hasbeen in the well for a prescribed time, the membrane and sorbentmaterial are forced together to draw off liquid before the next reagentis added.

Generally there is a dipstick test device for detecting an analyte in aliquid sample such as saliva or other biological fluid by treating theanalyte with at least one liquid reagent to form a detectable reactionproduct. The test device includes two components: a) means defining anaqueous permeable, aqueous insoluble reaction zone, adapted to retainthe detectable reaction product; and, integral with or separate from thereaction zone, b) a control absorbent above, and in liquid-transferringrelation with, the means defining a reaction zone. The control absorbenthas a predetermined, limited, liquid-absorbing capacity. The dipstick issized and configured for insertion in a vessel containing the sample,with the control absorbent oriented above the means defining a reactionzone, so that the control absorbent fills to capacity and the meansdefining a reaction zone incubates with the sample.

The reaction zone comprises at least one reactant e.g. a specificbinding partner for the analyte participating in a reaction to form thedetectable product.

The reaction product is detected by visual inspection, and the meansdefining a reaction zone is visible by external inspection of thedevice; optionally, the device includes a contrast region surroundingthe reaction zone to aid in the assay by contrasting with the reactionzone in respect to a characteristic being assayed; also optionally, thedevice can include an intensity scale for quantitative detection ofsample analyte.

The reaction zone is either integral with the control absorbent or it isattached to a face of the control absorbent. The reaction zone candefine at least two reaction regions, and the test device comprisesmeans for isolating the reaction regions from each other. At least onereaction region may be a control region.

The device includes an aqueous impermeable face plate having at leastone opening to allow liquid to reach the means defining a reaction zone.In order to provide a flush test head, the means defining a reactionzone comprises a flat reagent retention element having a node positionedto extend into each face plate opening.

The test may include a reagent pack sized and configured to supply aplurality of reagents to the reaction zone. For example, the reagentpack may include liquid reagents for generating a detectable reactionproduct.

The reaction zone can be positioned at one end of an elongated dipstick,and the device further can comprise a filter assembly positioned at anend of the dipstick. The device can include a reaction tray comprising awell for the filter assembly on the dipstick and to retain the filterassembly as the dipstick is removed from the well.

Detecting an analyte in a sample is by reacting the analyte with atleast one reagent to form a detectable reaction product. The method caninclude:

a) providing a test device comprising a control absorbent above, andin-transferring relation with, a defined a reaction zone, the controlabsorbent having a predetermined, limited, liquid-absorbing capacity;the control absorbent is in liquid-transferring relationship with thedefined reaction zone;b) inserting the dipstick into a vessel containing a predeterminedvolume of sample, with the control absorbent oriented above the definedreaction zone;c) incubating the predetermined sample volume with the reaction zone;d) allowing formation of the detectable reaction product; ande) detecting the reaction product.

Different Criteria for Dogs and Cats

For a healthy control dog or cat, the salivary IgA antibody levels aregenerally below about 10 U/ml (dog) or below about 25 μml (cat). Thesalivary IgM antibody levels are generally below about 25 U/ml forhealthy dog or cat.

For a patient dog or cat with moderate food allergy and foodintolerance, at the salivary IgA antibody levels are generally at about15 U/ml (dog) or at about 30 U/ml (cat). The salivary IgM antibodylevels are at about 35 μml for patient dog or cat.

For a patient dog or cat with severe food allergy and food intolerance,the salivary IgA antibody levels are generally below about 20 μml (dog)or about 35 μml (cat). The salivary IgM antibody levels are generally atabout 40 U/ml (dog or cat).

Most of the readings (about 98%) for the salivary IgA antibody levels inthe healthy control dog or cat are under about 10 U/ml (dog) or 25 U/ml(cat). Likewise, most of the readings (about 85%) for the salivary IgMantibody levels in the healthy control dog or cat are under about 25U/ml. However, there are certain readings in the panel of the healthycontrol dog or cat that are higher than about 10 U/ml (dog) or 25 U/ml(cat). Particularly higher readings in the healthy control dog or catmay indicate sensitivity to the corresponding dietary antigen.

In the patient dog or cat with moderate food allergy and foodintolerance, most of the readings for salivary IgA antibody levels areabove about 15 U/ml (dog) and 30 U/ml (cat). In the patient dog or catwith severe food allergy and food intolerance, most of the readings forsalivary IgA antibody levels are above 20 U/ml (dog) and 35 U/ml (cat).

The serum antibody can be IgA, IgM, IgG or immune complex. The serumlevel of at least one of:

(a) about 100 mg/dl (dog), and about 300 mg/dl (cat) of IgA, or(b) about 200 mg/dl (dog) and about 300 mg/dl (cat) of IgM, or(c) about 1750 mg/dl (dog) and about 2500 mg/dl (cat) of IgG; oror a relative increase in the level of immune complex is indicative ofat least mild insensitivity or intolerance.

Many different formats are possible for carrying the sample bodily fluidto the reaction zone. In some cases the bodily fluid is applied to anappropriate filter paper or other carrier material and the filter paperor other carrier material with that fluid sample is impregnated on andin the paper or other carrier material carrier and is then sent to alaboratory by any convenient means for analysis. The paper or othercarrier material including the sample may, for instance, in one partcontain saliva or other bodily fluid, and in another separate part therecan be serum. By using filter paper or other carrier material as thecarrier, it can be easy for an owner of a pet to simply mail a sample toa laboratory for appropriate testing of one or more antigens. The filterpaper or other carrier material can have one or more reaction zones fordifferent antigens. This carrier system of filter paper or other carriermaterial permits for a wholly or partly dehydrated sample to be carriedto a laboratory for subsequent processing, which can include a hydrationstep prior to analysis in an appropriate analyzer.

The next or final stage of testing relates to genetic, DNA/RNA ornutrigenomics testing. As such the disclosure is concerned withnutritional genomics or nutrigenomics and nutrigenetics. Such testing inthe staged testing procedure is applied generally after either one orboth the saliva mucosal fluid or serum testing has been completed, andthe results of that testing are inadequate, inconclusive, non-definitiveor the patient needs further specificity or accuracy in relation to thefood intolerances or sensitivity.

The disclosure includes a method of modulating the regulation of a geneor the protein expression or metabolites in an animal by nutritionalmanagement, including the step of analysing the gene or proteinexpressions or metabolites. Selected genes, proteins or metabolites inthe samples are identified for a particular phenotypic parameter. Theeffect of a biologically active nutrient varies for different genotypes.A biologically active nutrient is provided to the animal to modulate theselected genes, proteins or metabolites so as to change the response ofthe animal to the particular phenotypic parameter in a desirable manner.

Typical genes, proteins and metabolites are, for example, those involvedin the toxicology and nutrigenomics research (apoptosis, cell cycle, DNAdamage signalling pathway, drug metabolism phase I and phase II enzymes,PI3K-AKT signalling pathway, toxicology and drug resistance), cytokinesand inflammatory response (inflammatory cytokines and receptors,inflammatory response and autoimmunity, NFKβ signalling pathway, TNFligand and receptor), metabolic diseases (diabetes, insulin signallingpathway, obesity, oxidative stress and antioxidant defences) andneurological disorders (depression, epilepsy, general anxiety disordersand panic disorders).

The animals can be selected from livestock, companion, sporting, workingand different domesticated pet and laboratory animals, also includingfish. These can include for example the following: birds, cat, cattle,dog, donkey, goat, guinea pig, hamster, horse, mouse, pig, poultry,quail, parrots, rabbit, rat, salmon, sheep, trout and turkey or exoticanimals.

The phenotypic parameter can be, for example, growth, reproduction,lactation, maintenance, geriatric, inherited and acquired diseases,allergic, arthritic, autoimmune, inflammatory, metabolic andpathopsychological or psychological conditions.

The identification of the selected genes, proteins or metabolites in thesample can be effected by high throughput screening (HTS) techniques,such as microarray, pathway specific microarray, serial analysis of geneexpression and gene sequencing. Alternative HTS methods to analyse thesample include proteomic and metabolomic assays.

The term “healthy” is a well defined term. In this application the termrefers to an individual animal that has been determined to be well onthe basis of physical examination, laboratory data of blood or otherbiological fluids or tissues, and the information provided by theanimal's caregiver, owner or guardian.

The term “unhealthy” is a well defined term. In this application theterm refers to an individual animal with physical or physiological orpathological or genetic deviation from the state of health.

The term “biologically active nutrient” in this application refers to acompound or composition or ingredient of an ingested material that hassome biological measurable or documented effect in the body of anindividual animal.

The method includes identifying a biologically active nutrient based onwhat is termed the “molecular dietary signature” that the biologicallyactive nutrient induces in an individual animal, the molecular dietarysignature being a variation of expression of a set of genes, protein ormetabolites which may differ for the genotype of the individual animal.

The molecular dietary signature relates to the interaction between thenutritional environment and genome in an individual in the sense ofnutritional genomics or nutrigenomics. The basic concept is thatchemical nutrients affect gene expressions in a specific mode switchingfrom health to a pathophysiological condition or vice versa. Theadvancement of knowledge in human and animal genomes and the spread ofbiotechnology offer the opportunity to individualize dietaryintervention to prevent, mitigate or cure chronic diseases (i.e.,individualized nutrition). The concept applies not only to companion petanimals, laboratory animals, but also to nutrient-genome interactions infarm animals. For farm animals, nutrigenomics can be applied for theimprovement of productive performances, and the control of infectiousand metabolic diseases, through the use of appropriated dietarycompositions or supplements.

In companion pet animals, nutrigenomics can be directed to enhancementor maintenance of health and quality of life through the identificationof the most suitable diet or supplementation to maintain or optimize thephysiological health.

The animal genome and biotechnology systems, such as microarrayplatforms, can be used to modify the effect of nutrients on gene andprotein expression profiles and the adaptation of animals to nutrientexposure, and as a mechanism to identify genetic variants with favorableor unfavorable traits. Nutrigenomics, namely the integration offunctional genomics, nutrition, health and biological response, and theregulatory role of nutrients on gene expressions is enabled bymicroarray technology and integrated on an informatics platform.Nutrigenetics is the retrospective analysis of genetic variations amongindividuals with regard to their clinical response to specificnutrients.

The high throughput screening technologies are employed to identify alarge number of markers or target molecules of a specific parametertreatment or pathology. This is applied to animal or pet nutrition toidentify a set of genes, proteins, metabolites or other markers that areunique for a specific intake of each nutrient, chemical compound orxenobiotic. A specific nutrient affects body response in a form that isa molecular dietary signature.

This same concept as applied to gene expressions, measured withmicroarray technology, leads to the identification of a unique moleculardietary signature for each specific nutrient. In the case of geneexpressions, the utilization of a public data repository allows theidentification of a set of genes involved in biological processes,molecular function or cellular component, or in a mix of them, which areaffected by the dietary change or composition. The three mainclassifications of gene functions are incorporated in the gene ontologyproject, which provides a controlled vocabulary to describe gene andgene product attributes in any organism. Other classifications are(KEGG, Kyoto Encyclopaedia of Gene and Genomes; and Biocarta) toidentify the unique signature that a dietary change or composition isable to produce in an organism.

The signature of a particular nutrient can also vary from individual toindividual, according to the DNA polymorphisms of the genes or genome.In the case that the genetic make-up of the individuals is known, themolecular dietary signature of mutant animals compared to that ofwild-type animals forms a family of molecular signatures, which are usedfor the identification of the action of the nutrient.

Example

Compound A is an anti-arthrosis natural plant extract which is fed to agroup of 20 dogs, 10 healthy and 10 unhealthy dogs affected byarthrosis. The compound is fed for 15 days. Before and after the periodof administration, a blood sample is drawn and used for a transcriptomeanalysis (gene expression) using a commercial oligomicroarray containing44000 probes. The number of genes which significantly varied after thetreatment is 73, when compared to those of the group of healthy animalsthat received a placebo.

Data mining using a public domain repository database and softwareindicated that the 73-gene variation of gene expression involved theGene Ontology pathway response to stress, external stimuli, immunesystem process and cell communication. The average number of genesinvolved in each pathway is 15 (10 up-regulated and 5 down-regulated),10 (5 up-regulated and 5 down-regulated), 23 (18 up-regulated and 5down-regulated) and 25 (5 up-regulated and 20 down-regulated),respectively for a total of 73 genes (38 up-regulated and 35down-regulated). These genes form a distinct cluster molecular dietarysignature, which significantly differs from the level of expression ofthe placebo fed control group of dogs, and represent the action andresponse of the organism to the dietary compound. No other dietarycompounds tested will produce the same molecular dietary signature whenadministered to dogs.

Gene Ontology Up-regulated Down-regulated Total Response to stress 10 515 Response to external stimuli 5 5 10 Immune system process 18 5 23Cell communication 5 20 25 Total 38 35 73

However, in looking at the individual response for each dog of the groupreceiving Compound A, some variations occurred. In other words, if theaverage values are 38 genes up-regulated and 35 genes down-regulated,some of these genes will not change expression levels in some of thedogs receiving compound A. In the example, 5 of 10 dogs responddifferently to the dietary administration of compound A.

Gene Ontology Up-regulated Down-regulated Total Response to stress 8 513 Response to external stimuli 5 2 7 Immune system process 10 5 15 Cellcommunication 4 18 22 Total 27 30 57

In the example, genotyping of these dogs indicated that the 5individuals with a different response to the biologically activecompound A presented a single nucleotide polymorphism (SNP) of thecanine CYP1A2 gene that results in a deficiency of cytochrome P450activity. For the biologically active compound A, two molecular dietarysignatures are reported, one for each genotype.

There is a method of identifying a biologically active nutrient for anindividual animal having a genotype, which comprises:

(a) using a “reference” dataset containing functional genomic profilesof biological samples of the genotypes of different animals of thespecies, the different animals being healthy animals;(b) selecting a “target” dataset containing the functional genomicprofile of biological samples of the genotypes of different animals, thedifferent animals being unhealthy animals;(c) using a “biologically active nutrient” dataset comprising differenteffects of biologically active nutritional components on functionalgenomic profiles of the different animals of different genotypes fromthose of the target group (b), the different genotypes being differentlyresponsive to the same biologically active nutritional components; and(d) having the reference dataset or target dataset include an individualanimal for which the biologically active nutrient is to be identified.

At least one of the “reference” or “target group” datasets is relatedwith the “biologically active nutrient” dataset to identify abiologically active nutrient for the selected animal genotype toprevent, treat, control, or modulate a state of physiologicalhomeostasis or pathophysiological condition of the individual animal inthe reference dataset or target group.

The identification is based on the molecular dietary signature being theexpression of a gene or a set of genes which may differ for thegenotypes of different animals of the same species. The nutrientidentification includes the molecular dietary signature that thebiologically active nutrient induces in the individual animal.

The animal can be either a canine or a feline. The canine or feline isfrom the group consisting of one or more breed type, specific breed,chronological age, physiological age, activity level, healthy, andunhealthy.

The pathophysiological phenotypic conditions can be any one or moreexamples of any inherited or acquired diseases or conditions such asautoimmunity, anxiety, arthritis, depression, variable body conditionscore, immune suppression, inflammation, aural disease, skin, aging andbehavioral changes, cancer or neoplasia, cardiovascular disease, oculardisease, orthopedic disease, endocrine disease, hematogical disease,kidney disease, gastrointestinal disorders including inflammatory boweldisease (IBD), acute or chronic diarrhea, exocrine pancreaticinsufficiency, mal-digestion and pancreatitis, hepatic disorder, liverdisease, obesity, dental disease, and pulmonary disease.

The data of the individual animal can be one or more data items relatedto genotype, including breed, breed(s) of parents, pedigree, sex, coattype, and evident hereditary conditions and disorders. Physiologicalrelated conditions include one or more of age, weight, veterinarymedical history, reproductive history, health or unhealthy conditions,appetite, physical activity level, mental acuity, behavioralabnormalities and disposition.

The reference data can include one or more data of DNA, RNA, proteins,metabolites and biomarkers selected from an individual animal or groupsof animals with different genotypes in physiological homeostasis.

The target group data can include one or more data of DNA, RNA,proteins, metabolites and biomarkers selected from an individual animalor groups of animals with different genotypes in non-physiologicalhomeostasis.

The biologically active nutrient data can include one or more data ofDNA, RNA, proteins, metabolites and biomarkers selected from anindividual animal or groups of animals with different genotypes, thedifferent genotypes being responsive differently to the same nutritionalcomponents.

The data comprise analytical data from a biological sample obtained froman individual animal.

The identified nutrient can be one or more of a food, part of a food, asupplement, a nutraceutical or any biologically active nutrient selectedto enhance an aspect of health of an animal. Health can be promoted bypreventing, attenuating or eliminating at least one disease state in oneor more animals or by restoring physiological homeostasis.

Nutrigenomics is the identification of the appropriate nutrient tomodify the phenotype, based on nutrient-inducible genes, nutrigeneticsrepresents the identification of the appropriate nutrient for a definedgenotype. Nutrigenetics is an applied science, driven by the paradigmsof nutritional pharmacology, the onset of genetic polymorphism, and ofclinical experience. Nutrigenomics is a discovery science, driven by theparadigms of molecular biology, enabled by microarray technology, andintegrated on an informatics platform.

The role of gene-nutrient interaction is recognized for some monogenicand multi-factorial defects. Monogenic diseases are determined by asingle gene and multi-factorial diseases by the combination of severalgenes with other non-genetic factors. Sometimes, the classification maybe an oversimplification, since monogenic diseases also may involve morethan a single gene and environmental factors can modulate the expressionof phenotype. Some classical monogenic diseases in humans arephenylketunuria, galactosemia, lactose intolerance and celiac disease.In most of the case of monogenic disease, dietary intervention can beused to avoid or treat the patients. In the case of phenylketunuria, anautosomal recessive defect resulting from a deficiency of phenylalaninehydroxylase which leads to mental retardation, a phenylalaninerestricted diet avoids the severe consequences of the disease.Similarly, galactosemia, an autosomal defect, is related to thedeficiency of one of the three main enzymes involved in galactosemetabolism (galactose-1-phosphate uridyltransferase, galactokinase,uridine-diphosphate galactose-4′ epimerase), impairing galactosemetabolism, resulting in feeding difficulties, and prolonged conjugatedhyperbilirubinemia during neonatal life. Avoidance of breast feeding andgalactose in the diet prevent the consequences of this defect.

Among the multi-factorial chronic/age-related diseases, cardiovasculardiseases, and metabolic syndrome, cancer, osteoporosis and neurologicaldiseases are some classical examples in humans and these syndromes aregenerally associated with the aging process. Senescence is an obligatefate of cells, but gaining the knowledge of the gene-environmentinteractions can be effective in reducing the gap between normal andideal-healthy-aging.

Dietary factors are relevant for the onset and progression ofdegenerative diseases and solid scientific evidence has to be providedto support nutritional intervention. Also the multi-factorialchronic/age related diseases respond in a different way according to thegenotype of the individual animal, leading to a so-called “individualsusceptibility” or “genetic risk factor”.

The disclosure integrates the concepts of nutrigenetics with that ofnutrigenomics, considering:

(a) the different genetic make up of individual animals, or a group ofthem;(b) the different functional genomic profile for different phenotypicclasses of animals (namely healthy, unhealthy, affected, not affected,physiological states, pathophysiological conditions); and(c) the variable response of an individual animal or group of animals toa nutrient.

The “genotypic information” relates to genetic mapping, geneticbackground, and genetic screening databases. This includes data obtainedfrom the pedigree, family history, heritable physical characteristics,genetic screening tests, DNA testing, genomic mapping, and relatedlaboratory assessment of the gene product for known or suspectedcongenital and heritable traits. In this application, the term “geneproduct” means the specific phenotypic characteristic(s) resulting fromthe expression of the genotype, and may include certain specificlaboratory or other test data.

The “genotypic information” typically relates to individual animals, ora group or class of animals. This genotypic information, namely thephysical characteristics and genetic makeup (pedigree), heritabledisorder history, and related health history of animals in the group isusually manually recorded by breeders, owners, and researchers ofcompanion and other valued animals. The genetic constitution of anindividual includes genes without visible effects as well as thoserevealed by the phenotype. It may refer to all the genes or to a singlepair of alleles.

“Genotyping” refers to the process of determining the genotype of anindividual by the use of biological assay, such as polymerase chainreaction (PCR), DNA sequencing, and DNA microarrays. The techniqueprovides a measurement of the genetic variation between members of aspecies and is uses to investigate disease, productive, reproductive andnutrition-associated genes. The most common type of genetic variation isthe single nucleotide polymorphisms (SNP) that is a single base pairmutation at a specific locus, usually consisting of two alleles. SNPsare often found to be associated with many diseases, productive andreproductive traits of animals and are becoming of particular interestin pharmacogenetic, pharmacogenomic, nutrigenetic and nutrigenomicstudies.

A group of animals of the same specie having the same genotype includesindividuals that share a minimum number of common SNPs or other DNAmarkers that are related to a defined characteristic. In that sense, oneanimal can be included in several genotype groups, according to thespecific characteristic to which that the group relates.

In humans, the use of SNPs is being extended to the haplotype (HapMapproject), which is attempting to provide the minimal set of SNPs neededto genotype the human genome. Similar haplotyping is being extended toanimals.

SNPs can also provide a genetic fingerprint for use in identity testing.

The “group” can be defined at least in part by a physiological conditionthat is a product of interaction of the genotype with the environment ofan animal or a group of animals. The term “physiological condition”refers to one or more of the physical, behavioral and biochemicalattributes of an animal including its size, weight, age, sex, activitylevel, disposition, and condition of heath or disease.

“Functional Genomic Profile” as used in this disclosure includes DNAregions transcribed into RNA, expressed genes, expressed sequence tag(EST), micro RNA, translated proteins and their derived metabolites. Afunctional genomic profile can be established using any one or more of agenomic, proteomic or metabolomic approach. A functional genomic profilecan result from information from DNA, RNAs, peptides, proteins, ormetabolites associated with a phenotypic condition of an animal inresponse to exposure to one or more biologically active nutrients.

Information for the Functional genomic profile as used in thisdisclosure is generated from biological samples by any technique knownin the art of functional genomics. Examples of techniques useful ingenerating functional genomic analysis include, without limitation, thefollowing techniques that can be used individually or in combination:(a) DNA, cDNA, RNAs and protein arrays and microarrays in the existinglow and high density formats; (b) polymerase chain reaction (PCR)techniques including single and multiplexed quantitative real-time PCRtechniques; (c) serial analysis of gene expression (SAGE); (d) DNA andRNA sequencing; (e) Southern blot analysis, Northern blot analysis andWestern blot analysis; (f) gel electrophoresis, including two-color 2Dgel methodologies, SDS-polyacrylamide gel electrophoresis (SDS-PAGE),and 2D PAGE; (g) protein sequencing, using variable existing massspectrometry techniques; (h) metabolite analysis, using variableexisting mass spectrometry techniques; (i) liquid chromatography byitself or in tandem with mass spectrometry techniques and otherseparative analytical techniques.

As used in this disclosure, the functional genomic profile extendsbeyond measurements of clinical pathology analytes such as completeblood count, serum chemistry, hormone assays and analysis.

The functional genomic profile of an animal can be associated with a“normal” or “abnormal” phenotype. A “normal” phenotype is one occurringin an animal exhibiting a condition of health as defined herein, andgenerally indicative of such a state. A “normal” phenotype is associatedwith physiological homeostasis, i.e., a tendency to stability of optimalbodily functions. An “abnormal” phenotype is one that is outside therange identified as “normal” and can be associated with a breakdown inphysiological homeostasis or pathophysiological condition.

A functional genomic profile from a normal phenotype differs at least inone piece of data or information from the functional genomic profile ofan abnormal phenotype. A progressive drift from normality can lead tothe death of the individual, requiring an intervention to restore thephysiological homeostasis to a healthy, normal condition.

A normal phenotype can present a functional genomic profile generallyassociated with an abnormal phenotype, indicating a latentnon-physiological homeostasis or hereditary predisposition. This driftfrom the normality requires a preventive or prophylactic intervention torestore the physiological homeostasis to abnormal healthy condition.

“Biological samples” include for instance feces and urine, blood, lymph,tears, cheek swab, saliva, amniotic fluid, serum, prostatic and vaginalsecretions, hair, tissue biopsies and necropsy specimens.

The “reference dataset” includes the functional genomic profile ofbiological samples and genotype information for the animals with normalphenotype, typically stored in digital form and organized in one to aplurality of databases.

The “target group dataset” contains the functional genomic profile ofbiological samples and genotype information for the animals in abnormalunhealthy conditions.

The “nutrient dataset” comprises genotype information and the differenteffects of biologically active nutrients on a functional genomic profileof animal of different genotypes.

The different genotypes respond differently to the same nutritionalcomponents, and according to the present disclosure, effects ofbiologically active nutrients on the functional genomic profile can bedetermined by controlled experiments in animals having differentgenotypes and exposed to different levels of, and/or different durationsof exposure to, one or more biologically active nutrients.

In one embodiment, an alternative testing model of biologically activenutrients is an ex vivo model using tissue explants obtained from ananimal of the same species and the same genotypes, and maintainedoutside the body of the animal.

The nutrition data set can include data not only on chemical orbiological entities known as biologically active nutrients but on avariety of materials that have nutritional, or nutraceutical orpharmacological effect. All such materials are considered biologicallyactive nutrients herein if a useful effect on expression of at least onegene, function of at least one protein or production of at least onemetabolite is found. In one embodiment, biologically active nutrients ofinterest herein are materials having GRAS (generally regarded as safe)or equivalent status under U.S. FDA (Food and Drug Administration)regulations or counterpart regulations in other countries, or areeligible for such status. In other embodiments a biologically activenutrient can be a therapeutically or pharmacologically effectivecompound, e.g., a drug or herbal medicine.

Otherwise, the macronutrients required in a balanced animal diet(protein, carbohydrate, fat and fiber) are considered separately frombiologically active nutrients such as those listed above in designing anutritional formula, as will be discussed below.

Certain biological materials, especially botanical materials, can beconsidered biologically active nutrients and can, if desired, beincluded in the nutrition data set. In many of these, a bioactivechemical entity has been identified. Even where a bioactive component isknown, other unknown, bioactive components may be present and contributeto the bioactive effect or effects of the biological material.

Examples of macronutrients are set out:

MACRO-NUTRIENTS Chicken meat Beef meat Lamb meat Horse meat Turkey meatBison meat Ostrich or Enu meat Rabbit meat Venison meat Fish, Egg RiceCarrot Pumpkin Peas Beet, sugar pulp Soy hulls Potato Oats Oil,vegetable

Examples of micronutrients and biologically active nutrients are setout:

MICRO-NUTRIENTS AND BIOLOGICALLY ACTIVE NUTRIENTS Leucine IsoleucineValine Alanine Glutamine Taurine L-Carnitine Portulaca oleraceaAndrographis paniculata Butea frondosa Sylibum marianum Echinaceaangustifolia Curcuma longa Eleutherococcus senticosus Valerianaofficinalis Matricaria recutrita Conjugated linoleic acid Na sulphateGlucosamine HCl Vaccinum nirillus Vitamin E Vitamin C Vitamin B1 VitaminB2 Di-methylglycine g-orizanol EPA + DHA Green tea polyphenols

Data defines the genotype and physiological condition of the individualanimal for which a diet is designed, and a nutrition product orcomposition prepared. This includes the functional genomic profile. Inorder to design the nutritional formula, the input data for an animal iscompared with reference data set and target data set to identify thenormal or abnormal unhealthy conditions of the individual animal.

The nutrient data set contains the effects of biologically activenutrients on the functional genomic profile of an individual animal withdifferent genotypes. The nutritional formula is computed to incorporateeffective amounts of one or more biologically active nutrients accordingto the specific effects on the functional genomic profile in order torestore the physiological homeostasis. The nutritional formula can becomputed as a dietary or nutritional supplement which can be related to,exclude, or include basic energy, protein, metabolic or other nutrientrequirements.

Where a nutritional formula, food or composition is generated, thebiologically active nutrients and other components can be in anysuitable form. For example, components can be expressed in terms oftheir content in a food composition (e.g., in % or in mg/g, usually on adry matter basis), in terms of a daily dosage or allowance (e.g., ing/day), or optionally on a live weight basis (e.g., in mg/kg/day). Anillustrative nutritional formula, food or nutrient composition can beobtained by the present disclosure and can for instance include any oneor more of the exemplary macro-nutrients, micro-nutrients and/oradditives set out above. The food composition could be one or morebiologically active nutrient formulas selected from the exemplarymacro-nutrients, micro-nutrient and/or additives setout above and selfcontained and/or added as a “sprinkle” supplement in a dry liquid orsemi-moist form to an existing regular or specialized or therapeuticdiet.

Animals in conditions of health or disease are identified. Each sampleis subjected to functional genomic analysis, for example using anestablished microarray technique, to evaluate a functional genomicprofile for the animal that provided the sample, which reflects thegenotype, and physiological, and pathophysiological or other conditionof the animal at the time the sample was collected.

Biologically active nutrients are tested in one or more animal havingdifferent genotypes.

An example of the comprehensive diagnostic testing used in thisdisclosure are selected examples for diagnostic genetic panels, screensand microarray analysis or other High Through Put systems (“HTS”).

The databases of the functional genomic profile and the nutritional database can be used to assist in resolution of the disease state orcondition.

The system and procedure for carrying out the genetic test is, forinstance a microarray analysis of DNA or RNA. Thereafter there will bean analysis with a nutritional data base. This is be done by acomparison of the functional genomic profiles as necessary with one ormore of the nutritional databases. This disclosure utilizescomprehensive and cumulative data profiling in a novel way over time toallow one to predict the specific nutritional management interventionsthat will assist in the care and management of the very earliest stagesof specific abnormalities or trends that have been identified in thenutrition profile of animals, thereby extending and improving theirnutrition and longevity. This is a unique approach to scientifically andmedically determining by comprehensive and cumulative laboratoryprofiling of individual animals and animals within specified definedgroups to permit intervention in preventive and management and treatmentof general and veterinary medical nutrition care. Specifically, thisdisclosure directs the outcome of the laboratory profiling tonutritional and nutritional supplement management of the specificidentified abnormalities and trends over time to accomplish this goal.This is not only important but also practical because nutritionalintervention and management is relatively inexpensive, non-invasive andeasily accepted by the pet owner and the veterinary professional makingthese recommendations.

Food sensitivity and intolerance testing is used co-jointly indetermining a feeding regime. The method includes conducting a firsttesting for food sensitivity or intolerance diagnosis, such that whenreporting from a first test, a sensitivity or intolerance to aparticular food ingredient or composition, a first food without theingredient or composition from a first group of foods is prescribed.Usage of the first food is for a first short-time period.

Thereafter a second testing of the animal for food sensitivity orintolerance diagnosis is conducted. The second testing is based uponattenuation of the food sensitivity or intolerance diagnosis by removingthe particular food ingredient or composition that was found to bereactive. A second food without the identified particular ingredient orcomposition is selected from a second group of foods, and additionally abasal food can be prescribed to combine with the second food.

This is described in more detail.

Selected diets which are applicable to the food sensitivity andintolerance testing are arranged for instance in two phases. A dietaryand food regime is arranged around these phases.

Phase I Uses Cleansing or Therapeutic Diets

-   -   A. Testing: Perform salivary (and/or blood spot) P-O-S        diagnostic kit test, or serum testing or testing using heat map        DNA/RNA technology    -   B. Select Appropriate diet from 5 options of a first food group        is selected to be one of:        -   1. Liver/GI tract cleansing        -   2. Anti-Arthritic        -   3. Anti-Inflammatory        -   4. Anti-Allergy        -   5. Vegetarian (for meat protein allergic dogs)    -   Feed for 4-6 weeks or longer.    -   C. Repeat salivary (or blood spot) P-O-S test, or serum testing        or heat map testing. Should the sensitivity have been attenuated        the second food group can be used. This includes a different        formulation of a food product, for instance in sprinkles format.        These second group products are selected to be one of:        -   1. Liver/GI tract cleansing        -   2. Anti-Arthritic        -   3. Anti-Inflammatory        -   4. Anti-Allergy        -   5. Vegetarian (for meat protein allergic dogs)    -   Additionally a food which is a Basal Healthy Dog Diet is used.        Namely, this is in addition to the “sprinkles” formulation that        matches diets 1-5 in B.    -   In certain cases if there is no attenuation, namely a second        testing indicates that the B. option needs to be fed for a        second 4-6 weeks or longer. The feeding regime of B. is then        repeated.    -   D. Continue on Basal Healthy Dog Diet+matched sprinkle unless a        dog starts to be ill. In that event then retest according to A        and switch back to B. or change to C. for another 4-6 weeks or        longer.

Phase II Uses Special Needs Diets

-   -   E. Testing: Perform salivary (and/or blood spot) P-O-S        diagnostic kit test or serum testing using heat map DNA/RNA        technology. This is optional.    -   F. Select desired diet from 5 options:        -   1. Geriatric        -   2. Obesity        -   3. Immune Enhancement        -   4. Sport        -   5. Endurance    -   Feed for 4-6 weeks or longer then repeat Test E (optionally).    -   G. Thereafter a second food group is used. This includes a        different formulation of a food product, for instance in        sprinkles format. These second group products are selected to be        one of the foods for F.    -   Switch over to the sprinkle that matches diets F. 1-5.        Additionally a food which is a Basal Healthy Dog Diet is used.        Namely, this is in addition to the “sprinkles” formulation that        matches diets F. 1-5.    -   H. Continue on Basal Healthy Diet+matched sprinkle—unless dog is        doing poorly or becomes ill. If so then retest as in Test E. and        switch back to desired diet and sprinkle of F. or G.    -   I. Alternatively, the Phase I regime of cleansing diets for 4-6        weeks or longer program should be used and followed if needed.

In the specification, there have been disclosed typical preferredembodiments of the disclosure and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation. Some typical embodiments of the disclosure havebeen described. Many more examples, modifications and variations of thedisclosure are possible in light of the above teachings. For instance,although the disclosure and the claims indicate specific steps toperform the invention, the steps described are not limited to aparticular sequence of performance and in some circumstances two or moreof these steps could be undertaken simultaneously. It is therefore to beunderstood that within the scope of the appended claims the disclosuremay be practiced otherwise than as specifically described, and the scopeof the disclosure is set out in the claims.

1-14. (canceled)
 15. A method for diagnosing a food sensitivity in acanine comprising the steps of obtaining a sample of a serum, screeningthe sample to detect the presence of at least one of an IgA, IgM or IgGantibody or immune complex or immune complex with complement to aparticular food ingredient or composition, and diagnosing a foodsensitivity or intolerance based on the presence of the antibody orimmune complex or immune complex with complement.
 16. The method ofclaim 15 wherein the screening step utilizes an immunoassay, selectivelyan enzyme-linked immunosorbant assay (ELISA) testing system to detectthe presence of an antibody immune complex or immune complex withcomplement to a particular food ingredient or composition of foodingredients.
 17. The method of claim 15 wherein the antibody is IgA orIgM, and the level of about 15 U/mL (dog) of IgA is indicative ofsensitivity; or the level of 35 U/ml of IgM for dog is indicative ofsensitivity.
 18. The method of claim 15 wherein the food sensitivity isto at least one of wheat or other gluten, corn or soy, beef or othermeat or fish protein or dairy, eggs, grains, botanicals, oils from seedsor fish, vegetables, or fruit.
 19. The method of claim 15 wherein thelevel of at least one of: (a) about 100 mg/dl (dog), of IgA, or (b)about 200 mg/dl (dog), or (c) about 1750 mg/dl (dog); or or a relativeincrease in the level of immune complex or immune complex withcomplement is indicative of sensitivity.
 20. A method for diagnosing afood sensitivity in a canine comprising the steps of: collecting asample of saliva; screening the saliva to detect the presence of atleast one of IgA or IgM antibody immune complex or immune complex withcomplement to a particular food ingredient or composition; diagnosing afood sensitivity based on the presence of the antibody; collecting ablood sample; preparing serum from the sample; screening the serumsample to detect the semi-quantitative or quantitative presence of atleast one of an IgA, IgM or IgG antibody or immune complex or immunecomplex with complement to a particular food ingredient or composition;and diagnosing a food sensitivity based on the presence of the antibodyor immune complex or immune complex with complement.
 21. A method ofclaim 20 wherein the saliva screening step is divided into two stages,the first stage being a qualitative step to determine the sensitivityand a semi-quantitative or quantitative step.
 22. The method of claim 15wherein the food ingredient for which the method is performed iscontained in a composition being at least one of a preprocessed foodcomposition, balanced diet or recipe composition and including raw orcooked ingredients.
 23. The method of claim 20 wherein the foodingredient for which the method is performed is contained in acomposition being at least one of a preprocessed food composition,balanced diet or recipe composition and including raw or cookedingredients.
 24. The method of claim 15 including a multi-stage methodfor diagnosing a food sensitivity comprising determining a biologicallyactive nutrient in relation to the canine from a molecular dietarysignature, the molecular dietary signature for the canine being avariation of expression of a set of genes, proteins or metabolites whichmay differ for the genotype of each canine.
 25. The method of claim 20including a multi-stage method for diagnosing a food sensitivitycomprising determining a biologically active nutrient in relation to thecanine from a molecular dietary signature, the molecular dietarysignature for the canine being a variation of expression of a set ofgenes, proteins or metabolites which may differ for the genotype of eachcanine.
 26. A method for diagnosing a food sensitivity in an animalincluding a multi-stage method comprising obtaining a sample of asaliva; screening the sample of saliva to detect the presence of atleast one of IgA or IgM antibody to a particular food ingredient orcomposition, diagnosing a food sensitivity based on the presence of theantibody, immune complex or immune complex with complement, andobtaining a blood sample; preparing serum from the sample; screening theserum sample to detect the semi-quantitative or quantitative presence ofat least one of an IgA, IgM or IgG antibody, immune complex or immunecomplex with complement to a particular food ingredient or composition,and diagnosing an immunologic food sensitivity based on the presence ofthe antibody or immune complex, and determining a biologically activenutrient in relation to the animal from a molecular dietary signature,the molecular dietary signature for the animal being a variation ofexpression of a set of genes, proteins or metabolites which may differfor the genotype of each animal.
 27. A method as claimed in claim 26wherein the saliva screening step is divided into two stages, the firststage being a qualitative step to determine the sensitivity, and asemi-quantitative or quantitative step.
 28. The method of claim 26wherein determining a biologically active nutrient comprises: using a“reference” dataset containing the functional genomic profiles ofbiological samples for the multiple genotypes of different animals;using a “target group” dataset containing the functional genomicprofiles of biological samples for the multiple genotypes of differentanimals; using a “nutrient” dataset comprising the variable effects ofnutritional components on a functional genomic profile of an animal, thedifferent genotypes that are responsive differently to the samenutritional components; and relating the “reference” or “target group”datasets with the “nutrient” datasets, to derive a nutrient for theselected animal genotypes to prevent, or treat, or control, or modulatea state of physiological homeostasis or pathophysiological condition ofthe animal.
 29. The method of claim 15 including determining a feedingregime including conducting a first testing for food sensitivity orintolerance diagnosis, such that when reporting from a first test, asensitivity test to a particular food ingredient or composition isidentified, additionally prescribing a first food without the ingredientor composition from a first group of foods, prescribing usage of thefirst food for a first short time period, conducting thereafter a secondtesting of the animal for food sensitivity diagnosis, and reporting inthe second testing on the attenuation of the food sensitivity diagnosisto the particular food ingredient or composition, and then prescribing asecond food without the particular ingredient or composition, the secondfood being selected from a second group of foods, and then optionally oradditionally prescribing a basal food with the second food.
 30. Themethod of claim 20 including determining a feeding regime includingconducting a first testing for food sensitivity or intolerancediagnosis, such that when reporting from a first test, a sensitivitytest to a particular food ingredient or composition is identified,additionally prescribing a first food without the ingredient orcomposition from a first group of foods, prescribing usage of the firstfood for a first short time period, conducting thereafter a secondtesting of the animal for food sensitivity diagnosis, and reporting inthe second testing on the attenuation of the food sensitivity diagnosisto the particular food ingredient or composition, and then prescribing asecond food without the particular ingredient or composition, the secondfood being selected from a second group of foods, and then optionally oradditionally prescribing a basal food with the second food.
 31. Themethod of claim 26 including determining a feeding regime includingconducting a first testing for food sensitivity or intolerancediagnosis, such that when reporting from a first test, a sensitivitytest to a particular food ingredient or composition is identified,additionally prescribing a first food without the ingredient orcomposition from a first group of foods, prescribing usage of the firstfood for a first short time period, conducting thereafter a secondtesting of the animal for food sensitivity diagnosis, and reporting inthe second testing on the attenuation of the food sensitivity diagnosisto the particular food ingredient or composition, and then prescribing asecond food without the particular ingredient or composition, the secondfood being selected from a second group of foods, and then optionally oradditionally prescribing a basal food with the second food.
 32. Themethod of claim 20 wherein the antibody is IgA or IgM, and the level ofabout 15 U/ml (dog) of IgA is indicative of sensitivity; or the level of35 U/ml of IgM for dog is indicative of sensitivity.
 33. The method ofclaim 26 wherein the antibody is IgA or IgM, and the level of about 15U/ml (dog) of IgA is indicative of sensitivity; or the level of 35 U/mlof IgM for dog is indicative of sensitivity.
 34. The method of claim 15wherein the antibody is IgA or IgM, and the level of a level below about10 U/ml of IgA is indicative of tolerance; or a level below 25 U/ml ofIgM is indicative of tolerance.